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The influence of veterinary medicines on the decomposition of dung organic matter in soil
European Journal of Soil Biology 38 (2002) 155−159 www.elsevier.com/locate/ejsobi
The influence of veterinary medicines on the decomposition of dung organic matter in soil Christian Sommer a,*, Bo Martin Bibby b a
Department of Ecology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark b Department of Mathematics and Physics, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark Received 13 August 2000; accepted 3 May 2001
Abstract Six commonly used veterinary medicines were investigated for their influence on the decomposition of dung from treated cattle. Recommended doses of the antibacterial agents spiramycin and enrofloxacin, and of the antiparasitic compounds α-cypermethrin, fenbendazole, ivermectin and levamisole were given to heifers. Dung was collected prior to treatment and two days after treatment, and mesh bags with portions of 40 g of dung were placed in the soil. The organic matter was determined in mesh bags retrieved after 8, 12 and 16 weeks. When compared with dung from untreated cattle, the mean loss of organic matter was significantly lower in dung collected from heifers dosed with α-cypermethrin, fenbendazole, ivermectin, levamisole and spiramycin but not in dung from heifers treated with enrofloxacin. Ivermectin and spiramycin caused a reduced loss for all time intervals, i.e. for 0–8, 0–12 and 0–16 weeks. Levamisole and α-cypermethrin resulted in significant effects when measured over 0–12 and 0–16 weeks, whereas fenbendazole significantly reduced loss of organic matter for the time interval 0–16 weeks only. When an exponential decay model was applied to the data, all veterinary medicines, except spiramycin, resulted in significantly more organic matter remaining at infinite time, as estimated by the parameter b/a. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: α-Cypermethrin; Enrofloxacin; Fenbendazole; Ivermectin; Levamisole; Spiramycin
The terrestrial decomposer community may be exposed to residues of veterinary medicines when medicine is eliminated in faeces from treated animals and faeces are deposited in pastures and farm land. Provided that concentrations are sufficiently high, toxic effects may be expected from the residues of biocides such as antimicrobial and antiparasitic agents. Indeed, lethal and sublethal effects against insects living in dung pats have earlier been demonstrated when cattle are dosed with avermectins or synthetic pyrethroids, e.g. [5,7,12,16]. The use of veterinary medicines may also result in a reduced rate of dung pat
degradation, as shown in experiments where the organic matter was quantified in pats deposited on the soil surface [3,6,11]. Studies on the side effects of veterinary medicines have focused on a scenario in which grazing cattle are treated and residues are deposited in dung pats on the soil surface. Less attention has been devoted to potential side effects when stabled animals are treated and residues are deposited in the soil by application of dung or slurry. An obvious way to study the decomposition process is to quantify the dung organic matter in mesh bags at different times after these have been placed in the soil. This simple methodology was applied in the following experiment.
* Corresponding author. E-mail address: [email protected] (C. Sommer).
Six commonly used veterinary medicines indicated for the treatment of bacterial or parasitic infections in cattle were selected for examination of their influence on decom
1. Introduction
© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 1 1 6 4 - 5 5 6 3 ( 0 2 ) 0 1 1 3 8 - X
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position. Selection of the medicines was mainly based on their mode of excretion, i.e. faecal. A significant proportion of the three antiparasitic drugs ivermectin, levamisole and fenbendazole, and of the antibiotic spiramycin is excreted as parent drug in the faeces [1]. The use of α-cypermethrin has been demonstrated to result in toxic effects against dung insects [12], and the dung may therefore be expected to contain α-cypermethrin and/or metabolites of α-cypermethrin. ie. Although enrofloxacin (an antibiotic belonging to the group of flouroquinolones) is mainly excreted via the urine and only to a lesser extent via the faeces [10,15], but it was included in the study because flouroquinolones may degrade slowly [4].
2. Materials and methods 2.1. Treatment of calves, and collection and preparation of dung Twenty-four stabled Holstein Frisian or Red Dane heifers were used for the experiment. All heifers were fed an identical diet of wheat, peas, grass silage and barley straw. Estimates of weights were obtained by tape measurement of the trunk circumference, and the heifers were allocated into six groups to ensure approximately equal average body weight within groups (213–260 kg). Dung collected from the rectum of all heifers immediately prior to the application of veterinary medicines was mixed and served as negative control dung. The control dung was treated in the same manner as mentioned below for dung collected after treatment. Dosing of the six groups with four heifers each was as follows. Ten millilitres of α-cypermethrin (Flusat, 15 mg/ml) were applied as a single topical formulation along the back-line of four heifers. The flouroquinolone, enrofloxacin (Baytrilt, 100 mg/ml), was given by intramuscular injection at a dose of 1 ml/40 kg bw for three days. The macrolide, spiramycin (Spiravett, 100 mg/ml), was also given by intramuscular injection, but as a single dose of 1 ml/10 kg bw. One group received a benzimidazole, fenbendazole (Panacurt, 22%), in the form of granulate at an oral dose of 10 g per heifer. Ivermectin (Ivomect, 10 mg/ml), a macrocyclic lactone, was applied as a single subcutaneous injection of 1 ml/50 kg bw. Finally, a group of heifers was given a single dose of the pyrimidine, levamisole (Decarist, 75 mg/ml), intramuscularly at a dose of 1 ml/20 kg bw. The cattle that received α-cypermethrin were stabled separately to avoid any transfer of the pour-on formulation by physical contact with other heifers. Dung was collected from the rectum of all heifers on February 26, 1999, which was two days after (the last) dosing. Each type of dung was thoroughly mixed and kept at 5 °C for two days while dry matter was determined. Dry matter was then adjusted to 11% by addition of deionised water, and the dung was subsequently deep-frozen
at –29 °C. On the day prior to the beginning of the field experiment, 40 g portions of thawed dung were weighed out on nylon mesh netting cut into 7 × 7 cm2 squares. The mesh size was approximately 1 mm, i.e. large enough to allow the mesofauna access to the dung. Eighteen mesh bags were prepared for each dung type, but less dung was available from heifers treated with spiramycin, and the number of bags was limited to 14. Organic matter was determined in three subsamples from each type of dung by weight loss on ignition at 500 °C. 2.2. Study site The field experiment was performed at the farm Højbakkegård, Tåstrup, 20 km west of Copenhagen, Denmark, on a pasture that had been grazed by cattle and sheep for the last 12 years. In August 1999, a plot was fenced off and the grass was cut immediately before the mesh bags were placed in the soil on August 9, 1999. One-hundred and forty-four sites with an approximate distance of 0.5 m were arranged in a 12 × 12 square and marked with small plastic tags. Five centimetre deep holes were made by pressing a metal cylinder through the soil surface and removing turf and soil core. Each core was shortened to allow sufficient space for the mesh bag, so that the turf was level with the surrounding soil surface when the core and turf were replaced in the hole. One-hundred and twenty-two mesh bags were placed in the holes, leaving 22 void sites. The different types of dung were placed alternately, and care was taken to ensure an even distribution of dung types over the area. Six replicate mesh bags containing dung from each treatment group were removed after 8, 12, and 16 weeks. Four, five, and five mesh bags with dung from heifers treated with spiramycin were retrieved at weeks 8, 12, and 16, respectively. 2.3. Processing of samples The contents of each mesh bag were weighed and dried at 105 °C until constant weight. Organic matter was determined by weight loss on ignition at 500 °C. 2.4. Statistics A mixed linear model with the qualitative effects of time, treatment, and the interaction between time and treatment was applied to the data with the use of PROC MIXED [8]. Eight variance groups with separate variance parameters were used in order to reflect the variation in the data. All observations corresponding to time zero constituted one variance group. For each dung type, a separate variance parameter was used for observations corresponding to time points greater than zero. In each group, variance homogeneity was determined by Bartlett’s test. Mean losses of dung organic matter for the untreated control group and each of the groups given a veterinary medicine were compared for
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157
Table 1 Mean loss of dung organic matter (mg/day) together with k and b/a estimated from an exponential decay model. Significance levels are given for comparisons between control dung from untreated heifers and dung from heifers treated with each of the veterinary medicines
Control Spiramycin Ivermectin Levamisole α-Cypermethrin Fenbendazole Enrofloxacin
Linear model
Exponential model
Loss of organic matter (mg/day) measured over time interval in weeks
Parameter
0–8
0–12
0–16
k
b/a
23.8 18.8 * 20.3 * 21.1 (n.s.) 21.7 (n.s.) 22.5 (n.s.) 24.9 (n.s.)
18.6 14.5 *** 15.5 *** 15.3 *** 15.2 *** 17.0 (n.s.) 17.3 (n.s.)
15.3 12.3 *** 13.1 * 12.1 *** 13.5 * 13.2 ** 13.8 (n.s.)
0.156 0.143 (n.s.) 0.153 (n.s.) 0.248 (n.s.) 0.176 (n.s.) 0.219 (n.s.) 0.286 (n.s.)
0.526 0.612 (n.s.) 0.616 * 0.620 * 0.620 * 0.626 ** 0.627 **
n.s.: Non-significant. * P < 0.05. ** P < 0.01. *** P < 0.001.
the time intervals 0–8, 0–12 and 0–16 weeks. This was done using approximate t-tests (an ESTIMATE-statement for the effect of interaction between time and treatment [8]). With the aid of PROC NLMIXED [9], an exponential decay model, based on first-order kinetics and variance groups identical to the above-mentioned ones, was used for additional analysis of the data. With OM(t), a and b denoting the amount of organic matter at times t, 0, and ∞, respectively, OM(t) = b + (a – b) · exp(–k · t). The rate constant, k, given by OM'(t)/(b – OM(t)), was estimated together with b/a, i.e. the amount of organic matter at t = ∞ relative to amount of organic matter at t = 0. Comparisons of k and b/a between control dung and dung from groups given a veterinary medicine were done by approximate t-tests. The two models were compared by a likelihood ratio test. 3. Results All 122 mesh bags were removed intact from the field plot and transferred to the laboratory. Five mesh bags were excluded from analyses, because a juvenile earthworm (length <1.5 cm) and a small cast were found in two bags and the contents of another three bags had a distinct yellow–brown, dry-rotten appearance. The five observations were: levamisole, α-cypermethrin and enrofloxacin at week 12, and levamisole and fenbendazole at week 16. Four samples, i.e. both samples in which an earthworm was found and two of the yellow–brown samples, also represented obvious statistical outliers (with less organic matter) as the group variances were not homogeneous (Bartlett’s test; P < 0.05), when the observations were included. Inclusion of the fifth data point that did not represent an outlier in a statistical sense (levamisole at week 16) did not change the result of the analyses of the data.
Mean loss of organic matter was significantly lower in dung collected from heifers dosed with spiramycin, ivermectin, levamisole, α-cypermethrin and fenbendazole (Table 1). The use of spiramycin and ivermectin caused the most pronounced effects, as evidenced by significant effects for all time intervals. The decomposition of dung from heifers treated with levamisole and α-cypermethrin was significantly reduced when measured over 12 and 16 weeks, whereas fenbendazole resulted in a significantly reduced loss of organic matter for the time interval 0–16 weeks only. No effects were detected in dung from heifers that were given enrofloxacin. When data were analysed by the exponential decay model, all veterinary medicines, except spiramycin, resulted in significantly higher estimates for b/a, i.e. organic matter remaining at infinite time relative to organic matter at time zero. This is reflected in Fig. 1. The non-significant effect of spiramycin may be explained by a high variation in organic matter after 12 and 16 weeks. Although the rate constant, k, for decomposition of the different dung types varied, the k values for the decomposition of dung from heifers given veterinary medicines were not significantly different (P > 0.05) from the k value for control dung. The exponential decay model and the linear model described the data equally well (likelihood ratio test; P = 0.13).
4. Discussion The experiment indicates that commonly used veterinary antibacterial and antiparasitic drugs negatively influence the decomposition of dung organic matter in soil. This was supported by analysis of loss of organic matter up to 16 weeks after deposition of dung in the soil and by estimates of b/a from the exponential decrease in organic
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Fig. 1. Exponential decay model applied to the amount of organic matter in mesh bags. Mesh bags contained dung from untreated control heifers and dung from heifers dosed with a veterinary medicine. The bags were placed in soil at depth of 5 cm for 8, 12 or 16 weeks.
matter over time. No significant difference in the rate constant, k, was found. However, the rate constant k (= OM'(t)/(b – OM(t)) was of less interest as it is a relative measure dependent on both b and organic matter at times t. The rate constant may, therefore, be high, as reflected by an initial rapid decline in organic matter (e.g. enrofloxacin, Fig. 1), but more organic matter may nevertheless remain at later times as estimated by b/a (Table 1). Although the study did not show whether the effects on decomposition were mediated by an activity against detritivores, microbial decomposers or both, cautious explanations may be inferred from earlier experiments. Residues of ivermectin and of α-cypermethrin have been found to be toxic against the insect fauna in dung pats deposited on the soil surface [5,6,11,12], which implies that detritivores living in the soil/dung are affected. Also, fungicidal activity may be anticipated from dung residues of fenbendazole, as other benzimidazoles are specifically used as commercial
fungicides. This is supported by observations of delayed decomposition of dung pats in the field (T.S. Svendsen, personal communication), although dung residues of fenbendazole are apparently non-toxic to dung insects [13] and earthworms [14]. Finally, spiramycin may reduce microbial respiration, whereas the influence of levamisole on decomposition may, to some extent, be mediated by a nematicidal property of dung residues [2]. In this study, dung was collected two days after treatment of stabled cattle, i.e. when maximum faecal concentrations were expected. Under practical conditions, effects on decomposition may be less pronounced, as lower residue concentrations will result from mixing with dung from untreated animals. Also, residues may degrade during the storage of dung prior to its deposition to soil. Further studies on the decomposition and N mineralisation are warranted to assess the implications of the practical use of veterinary medicines when animal excreta are incorporated into soil.
C. Sommer, B.M. Bibby / Eur. J. Soil Biol. 38 (2002) 155–159
Acknowledgements Thanks are due to K. Freiesleben for practical assistance and for access to the heifers, J. Olesen for help with the field experiment and H. Lynge Kristensen for technical assistance. Højbakkegård and S.M. Thamsborg are also thanked for allowing us to use the field plot.
References [1]
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H.R. Adams (Ed.), Veterinary Pharmacology and Therapeutics, seventh ed., Iowa State University Press, Ames, IO, 1995. D. Barth, E.M. Heinze-Mutz, W. Langholff, R.A. Roncalli, D. Schlüter, Colonisation and degradation of dung pats after subcutaneous treatment of cattle with ivermectin or levamisole, Appl. Parasitol. 35 (1994) 277–293. I.R. Dadour, D.F. Cook, C. Neesam, Dispersal of dung containing ivermectin in the field by Onthophagus taurus (Coleoptera: Scarabaeidae), Bull. Entomol. Res. 89 (1999) 119–123. H. Hektonen, V. Berge, V. Hormazabal, M. Yndestad, Persistence of antibacterial agents in marine sediments, Aquaculture 133 (1995) 175–184. R. Herd, L. Strong, K. Wardhaugh (Eds.), Environmental Impact of Avermectin Usage in Livestock, Vet. Parasitol. 48 (1993) 1–344 (special issue). M. Madsen, B. Overgaard Nielsen, P. Holter, O.C. Pedersen, J. Brøchner Jespersen, K.-M. Vagn Jensen, J. Grønvold, P. Nansen, Treating cattle with ivermectin: effects on the fauna and decomposition of dung pats, J. Appl. Ecol. 27 (1990) 1–15. T.J. Ridsdill-Smith, Survival and reproduction of Musca vetustissima Walker (Diptera: Muscidae) and a scarabaeine dung beetle in dung of cattle treated with avermectin B1, J. Aust. Entomol. Soc. 27 (1988) 175–178.
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[10] M. Scheer, Concentrations of active ingredient in the serum and in tissues after oral and parenteral administration of Baytril, Vet. Med. Res. 2 (1987) 104–118. [11]
C. Sommer, B. Steffansen, B.O. Nielsen, J. Grønvold, K.-M. Vagn-Jensen, J.B. Jespersen, J. Springborg, P. Nansen, Ivermectin excreted in cattle dung after subcutaneous injection or pour-on treatment: concentrations and impact on dung fauna, Bull. Entomol. Res. 82 (1992) 257–264.
[12] C. Sommer, K.-M. Vagn Jensen, J.B. Jespersen, Topical treatment of calves with synthetic pyrethroids: effects on the non-target dung fly Neomyia cornicina (Diptera: Muscidae), Bull. Entomol. Res 91 (2001) 131–137. [13] L. Strong, R. Wall, A. Woolford, D. Djeddour, The effect of faecally excreted ivermectin and fenbendazole on the insect colonisation of cattle dung following the oral administration of sustained release boluses, Vet. Parasitol. 62 (1996) 253–266. [14] T.S. Svendsen, C. Sommer, P. Holter, J. Grønvold, Survival and growth of Lumbricus terrestris fed on dung from cattle given sustained-release boluses of ivermectin and fenbendazole, Eur. J. Soil. Biol 38 (3) (2002). [15] P.M. Vancutsem, J.G. Babish, W.S. Schwark, The flouroquinolone antimicrobials: activity, pharmacokinetics, clinical use in domestic animals and toxicity, Cornell Vet. 80 (1990) 173–186. [16] K.G. Wardhaugh, B.C. Longstaff, M.J. Lacey, Effects of residues of deltamethrin in cattle dung on the development and survival of three species of dung-breeding insects, Aust. Vet. J. 76 (1998) 273–280.
The influence of veterinary medicines on the decomposition of dung organic matter in soil Christian Sommer a,*, Bo Martin Bibby b a
Department of Ecology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark b Department of Mathematics and Physics, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark Received 13 August 2000; accepted 3 May 2001
Abstract Six commonly used veterinary medicines were investigated for their influence on the decomposition of dung from treated cattle. Recommended doses of the antibacterial agents spiramycin and enrofloxacin, and of the antiparasitic compounds α-cypermethrin, fenbendazole, ivermectin and levamisole were given to heifers. Dung was collected prior to treatment and two days after treatment, and mesh bags with portions of 40 g of dung were placed in the soil. The organic matter was determined in mesh bags retrieved after 8, 12 and 16 weeks. When compared with dung from untreated cattle, the mean loss of organic matter was significantly lower in dung collected from heifers dosed with α-cypermethrin, fenbendazole, ivermectin, levamisole and spiramycin but not in dung from heifers treated with enrofloxacin. Ivermectin and spiramycin caused a reduced loss for all time intervals, i.e. for 0–8, 0–12 and 0–16 weeks. Levamisole and α-cypermethrin resulted in significant effects when measured over 0–12 and 0–16 weeks, whereas fenbendazole significantly reduced loss of organic matter for the time interval 0–16 weeks only. When an exponential decay model was applied to the data, all veterinary medicines, except spiramycin, resulted in significantly more organic matter remaining at infinite time, as estimated by the parameter b/a. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: α-Cypermethrin; Enrofloxacin; Fenbendazole; Ivermectin; Levamisole; Spiramycin
The terrestrial decomposer community may be exposed to residues of veterinary medicines when medicine is eliminated in faeces from treated animals and faeces are deposited in pastures and farm land. Provided that concentrations are sufficiently high, toxic effects may be expected from the residues of biocides such as antimicrobial and antiparasitic agents. Indeed, lethal and sublethal effects against insects living in dung pats have earlier been demonstrated when cattle are dosed with avermectins or synthetic pyrethroids, e.g. [5,7,12,16]. The use of veterinary medicines may also result in a reduced rate of dung pat
degradation, as shown in experiments where the organic matter was quantified in pats deposited on the soil surface [3,6,11]. Studies on the side effects of veterinary medicines have focused on a scenario in which grazing cattle are treated and residues are deposited in dung pats on the soil surface. Less attention has been devoted to potential side effects when stabled animals are treated and residues are deposited in the soil by application of dung or slurry. An obvious way to study the decomposition process is to quantify the dung organic matter in mesh bags at different times after these have been placed in the soil. This simple methodology was applied in the following experiment.
* Corresponding author. E-mail address: [email protected] (C. Sommer).
Six commonly used veterinary medicines indicated for the treatment of bacterial or parasitic infections in cattle were selected for examination of their influence on decom
1. Introduction
© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 1 1 6 4 - 5 5 6 3 ( 0 2 ) 0 1 1 3 8 - X
156
C. Sommer, B.M. Bibby / Eur. J. Soil Biol. 38 (2002) 155–159
position. Selection of the medicines was mainly based on their mode of excretion, i.e. faecal. A significant proportion of the three antiparasitic drugs ivermectin, levamisole and fenbendazole, and of the antibiotic spiramycin is excreted as parent drug in the faeces [1]. The use of α-cypermethrin has been demonstrated to result in toxic effects against dung insects [12], and the dung may therefore be expected to contain α-cypermethrin and/or metabolites of α-cypermethrin. ie. Although enrofloxacin (an antibiotic belonging to the group of flouroquinolones) is mainly excreted via the urine and only to a lesser extent via the faeces [10,15], but it was included in the study because flouroquinolones may degrade slowly [4].
2. Materials and methods 2.1. Treatment of calves, and collection and preparation of dung Twenty-four stabled Holstein Frisian or Red Dane heifers were used for the experiment. All heifers were fed an identical diet of wheat, peas, grass silage and barley straw. Estimates of weights were obtained by tape measurement of the trunk circumference, and the heifers were allocated into six groups to ensure approximately equal average body weight within groups (213–260 kg). Dung collected from the rectum of all heifers immediately prior to the application of veterinary medicines was mixed and served as negative control dung. The control dung was treated in the same manner as mentioned below for dung collected after treatment. Dosing of the six groups with four heifers each was as follows. Ten millilitres of α-cypermethrin (Flusat, 15 mg/ml) were applied as a single topical formulation along the back-line of four heifers. The flouroquinolone, enrofloxacin (Baytrilt, 100 mg/ml), was given by intramuscular injection at a dose of 1 ml/40 kg bw for three days. The macrolide, spiramycin (Spiravett, 100 mg/ml), was also given by intramuscular injection, but as a single dose of 1 ml/10 kg bw. One group received a benzimidazole, fenbendazole (Panacurt, 22%), in the form of granulate at an oral dose of 10 g per heifer. Ivermectin (Ivomect, 10 mg/ml), a macrocyclic lactone, was applied as a single subcutaneous injection of 1 ml/50 kg bw. Finally, a group of heifers was given a single dose of the pyrimidine, levamisole (Decarist, 75 mg/ml), intramuscularly at a dose of 1 ml/20 kg bw. The cattle that received α-cypermethrin were stabled separately to avoid any transfer of the pour-on formulation by physical contact with other heifers. Dung was collected from the rectum of all heifers on February 26, 1999, which was two days after (the last) dosing. Each type of dung was thoroughly mixed and kept at 5 °C for two days while dry matter was determined. Dry matter was then adjusted to 11% by addition of deionised water, and the dung was subsequently deep-frozen
at –29 °C. On the day prior to the beginning of the field experiment, 40 g portions of thawed dung were weighed out on nylon mesh netting cut into 7 × 7 cm2 squares. The mesh size was approximately 1 mm, i.e. large enough to allow the mesofauna access to the dung. Eighteen mesh bags were prepared for each dung type, but less dung was available from heifers treated with spiramycin, and the number of bags was limited to 14. Organic matter was determined in three subsamples from each type of dung by weight loss on ignition at 500 °C. 2.2. Study site The field experiment was performed at the farm Højbakkegård, Tåstrup, 20 km west of Copenhagen, Denmark, on a pasture that had been grazed by cattle and sheep for the last 12 years. In August 1999, a plot was fenced off and the grass was cut immediately before the mesh bags were placed in the soil on August 9, 1999. One-hundred and forty-four sites with an approximate distance of 0.5 m were arranged in a 12 × 12 square and marked with small plastic tags. Five centimetre deep holes were made by pressing a metal cylinder through the soil surface and removing turf and soil core. Each core was shortened to allow sufficient space for the mesh bag, so that the turf was level with the surrounding soil surface when the core and turf were replaced in the hole. One-hundred and twenty-two mesh bags were placed in the holes, leaving 22 void sites. The different types of dung were placed alternately, and care was taken to ensure an even distribution of dung types over the area. Six replicate mesh bags containing dung from each treatment group were removed after 8, 12, and 16 weeks. Four, five, and five mesh bags with dung from heifers treated with spiramycin were retrieved at weeks 8, 12, and 16, respectively. 2.3. Processing of samples The contents of each mesh bag were weighed and dried at 105 °C until constant weight. Organic matter was determined by weight loss on ignition at 500 °C. 2.4. Statistics A mixed linear model with the qualitative effects of time, treatment, and the interaction between time and treatment was applied to the data with the use of PROC MIXED [8]. Eight variance groups with separate variance parameters were used in order to reflect the variation in the data. All observations corresponding to time zero constituted one variance group. For each dung type, a separate variance parameter was used for observations corresponding to time points greater than zero. In each group, variance homogeneity was determined by Bartlett’s test. Mean losses of dung organic matter for the untreated control group and each of the groups given a veterinary medicine were compared for
C. Sommer, B.M. Bibby / Eur. J. Soil Biol. 38 (2002) 155–159
157
Table 1 Mean loss of dung organic matter (mg/day) together with k and b/a estimated from an exponential decay model. Significance levels are given for comparisons between control dung from untreated heifers and dung from heifers treated with each of the veterinary medicines
Control Spiramycin Ivermectin Levamisole α-Cypermethrin Fenbendazole Enrofloxacin
Linear model
Exponential model
Loss of organic matter (mg/day) measured over time interval in weeks
Parameter
0–8
0–12
0–16
k
b/a
23.8 18.8 * 20.3 * 21.1 (n.s.) 21.7 (n.s.) 22.5 (n.s.) 24.9 (n.s.)
18.6 14.5 *** 15.5 *** 15.3 *** 15.2 *** 17.0 (n.s.) 17.3 (n.s.)
15.3 12.3 *** 13.1 * 12.1 *** 13.5 * 13.2 ** 13.8 (n.s.)
0.156 0.143 (n.s.) 0.153 (n.s.) 0.248 (n.s.) 0.176 (n.s.) 0.219 (n.s.) 0.286 (n.s.)
0.526 0.612 (n.s.) 0.616 * 0.620 * 0.620 * 0.626 ** 0.627 **
n.s.: Non-significant. * P < 0.05. ** P < 0.01. *** P < 0.001.
the time intervals 0–8, 0–12 and 0–16 weeks. This was done using approximate t-tests (an ESTIMATE-statement for the effect of interaction between time and treatment [8]). With the aid of PROC NLMIXED [9], an exponential decay model, based on first-order kinetics and variance groups identical to the above-mentioned ones, was used for additional analysis of the data. With OM(t), a and b denoting the amount of organic matter at times t, 0, and ∞, respectively, OM(t) = b + (a – b) · exp(–k · t). The rate constant, k, given by OM'(t)/(b – OM(t)), was estimated together with b/a, i.e. the amount of organic matter at t = ∞ relative to amount of organic matter at t = 0. Comparisons of k and b/a between control dung and dung from groups given a veterinary medicine were done by approximate t-tests. The two models were compared by a likelihood ratio test. 3. Results All 122 mesh bags were removed intact from the field plot and transferred to the laboratory. Five mesh bags were excluded from analyses, because a juvenile earthworm (length <1.5 cm) and a small cast were found in two bags and the contents of another three bags had a distinct yellow–brown, dry-rotten appearance. The five observations were: levamisole, α-cypermethrin and enrofloxacin at week 12, and levamisole and fenbendazole at week 16. Four samples, i.e. both samples in which an earthworm was found and two of the yellow–brown samples, also represented obvious statistical outliers (with less organic matter) as the group variances were not homogeneous (Bartlett’s test; P < 0.05), when the observations were included. Inclusion of the fifth data point that did not represent an outlier in a statistical sense (levamisole at week 16) did not change the result of the analyses of the data.
Mean loss of organic matter was significantly lower in dung collected from heifers dosed with spiramycin, ivermectin, levamisole, α-cypermethrin and fenbendazole (Table 1). The use of spiramycin and ivermectin caused the most pronounced effects, as evidenced by significant effects for all time intervals. The decomposition of dung from heifers treated with levamisole and α-cypermethrin was significantly reduced when measured over 12 and 16 weeks, whereas fenbendazole resulted in a significantly reduced loss of organic matter for the time interval 0–16 weeks only. No effects were detected in dung from heifers that were given enrofloxacin. When data were analysed by the exponential decay model, all veterinary medicines, except spiramycin, resulted in significantly higher estimates for b/a, i.e. organic matter remaining at infinite time relative to organic matter at time zero. This is reflected in Fig. 1. The non-significant effect of spiramycin may be explained by a high variation in organic matter after 12 and 16 weeks. Although the rate constant, k, for decomposition of the different dung types varied, the k values for the decomposition of dung from heifers given veterinary medicines were not significantly different (P > 0.05) from the k value for control dung. The exponential decay model and the linear model described the data equally well (likelihood ratio test; P = 0.13).
4. Discussion The experiment indicates that commonly used veterinary antibacterial and antiparasitic drugs negatively influence the decomposition of dung organic matter in soil. This was supported by analysis of loss of organic matter up to 16 weeks after deposition of dung in the soil and by estimates of b/a from the exponential decrease in organic
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Fig. 1. Exponential decay model applied to the amount of organic matter in mesh bags. Mesh bags contained dung from untreated control heifers and dung from heifers dosed with a veterinary medicine. The bags were placed in soil at depth of 5 cm for 8, 12 or 16 weeks.
matter over time. No significant difference in the rate constant, k, was found. However, the rate constant k (= OM'(t)/(b – OM(t)) was of less interest as it is a relative measure dependent on both b and organic matter at times t. The rate constant may, therefore, be high, as reflected by an initial rapid decline in organic matter (e.g. enrofloxacin, Fig. 1), but more organic matter may nevertheless remain at later times as estimated by b/a (Table 1). Although the study did not show whether the effects on decomposition were mediated by an activity against detritivores, microbial decomposers or both, cautious explanations may be inferred from earlier experiments. Residues of ivermectin and of α-cypermethrin have been found to be toxic against the insect fauna in dung pats deposited on the soil surface [5,6,11,12], which implies that detritivores living in the soil/dung are affected. Also, fungicidal activity may be anticipated from dung residues of fenbendazole, as other benzimidazoles are specifically used as commercial
fungicides. This is supported by observations of delayed decomposition of dung pats in the field (T.S. Svendsen, personal communication), although dung residues of fenbendazole are apparently non-toxic to dung insects [13] and earthworms [14]. Finally, spiramycin may reduce microbial respiration, whereas the influence of levamisole on decomposition may, to some extent, be mediated by a nematicidal property of dung residues [2]. In this study, dung was collected two days after treatment of stabled cattle, i.e. when maximum faecal concentrations were expected. Under practical conditions, effects on decomposition may be less pronounced, as lower residue concentrations will result from mixing with dung from untreated animals. Also, residues may degrade during the storage of dung prior to its deposition to soil. Further studies on the decomposition and N mineralisation are warranted to assess the implications of the practical use of veterinary medicines when animal excreta are incorporated into soil.
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Acknowledgements Thanks are due to K. Freiesleben for practical assistance and for access to the heifers, J. Olesen for help with the field experiment and H. Lynge Kristensen for technical assistance. Højbakkegård and S.M. Thamsborg are also thanked for allowing us to use the field plot.
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